Roles of Chaperone/Usher Pathways of Yersinia pestis in a Murine
Model of Plague and Adhesion to Host Cells
Matthew Hatkoff,a,bLisa M. Runco,cCeline Pujol,a,b* Indralatha Jayatilaka,a,dMartha B. Furie,a,dJames B. Bliska,a,band
David G. Thanassia,b
Center for Infectious Diseases,aDepartment of Molecular Genetics and Microbiology,band Department of Pathology,dStony Brook University, Stony Brook, New York,
USA, and Department of Life Sciences, New York Institute of Technology, Old Westbury, New York, USAc
Yersinia pestis and many other Gram-negative pathogenic bacteria use the chaperone/usher (CU) pathway to assemble viru-
lence-associated surface fibers termed pili or fimbriae. Y. pestis has two well-characterized CU pathways: the caf genes coding for
the F1 capsule and the psa genes coding for the pH 6 antigen. The Y. pestis genome contains additional CU pathways that are
structed deletion mutations in the usher genes for six of the additional Y. pestis CU pathways. The wild-type (WT) and usher
pestisstrainscontainingdeletionsinCUpathways y0348-0352,y1858-1862,andy1869-1873 wereattenuatedforvirulencecom-
during pneumonic plague. We examined binding of the Y. pestis WT and usher deletion strains to A549 human lung epithelial
cells, HEp-2 human cervical epithelial cells, and primary human and murine macrophages. Y. pestis CU pathways y0348-0352
andy1858-1862 werefoundtocontributetoadhesiontoallhostcellstested,whereaspathway y1869-1873 wasspecificforbind-
mutants identifies three of the additional CU pathways of Y. pestis as mediating interactions with host cells that are important
is maintained in a number of natural reservoirs, including rats,
are primarily spread through the bite of infected arthropods, in-
cluding, most notably, the rat flea, Xenopsylla cheopis (37, 38, 74).
Y. pestis introduced into a human host through the bite of an
infected flea can enter the bloodstream and spread to a regional
and initiate an inflammatory response. This inflammation causes
the lymph node to swell and transform into a painful black bubo,
the hallmark of bubonic plague (53). Y. pestis can re-enter the
bloodstream from the lymph node, leading to septicemic plague
by this route results in a secondary pneumonic plague, which can
then lead to direct human-to-human spread of the bacteria via
respiratory droplets. Direct inhalation of Y. pestis results in a pri-
mary pneumonic plague. Humans infected with all forms of
vention (14, 53).
Y. pestis evolved from the enteric pathogen Y. pseudotubercu-
losis 1,500 to 20,000 years ago (2). Key to its adaptation to the
vector-borne lifestyle was the acquisition of two Y. pestis-specific
plasmids, pMT1 and pPCP1 (9). The pPCP1 plasmid encodes the
Pla plasminogen activating protease, which is important for the
ability of Y. pestis to proliferate and persist within the lungs in
pneumonic form and for its dissemination from the site of infec-
tion in bubonic form (9, 45, 62). Pla also contributes to the bac-
teria’s ability to adhere to and invade host cells, making it one of
the adhesins expressed by Y. pestis (4, 18). The second pestis-spe-
cific plasmid, pMT1, contributes to both transmission through
ersinia pestis is a facultative intracellular, Gram-negative bac-
terial pathogen that causes the deadly disease plague. Y. pestis
fleas and increased virulence in the human host. The caf genes
present on pMT1 code for expression of the fraction 1 (F1) cap-
sule. The F1 capsule, which is expressed at 37°C, is a major pro-
tective antigen of Y. pestis and forms a dense coating around the
vector and contributes to pathogenesis in the mammalian host
(61, 73). Y. pestis also contains a third plasmid, pCD1, that is
shared with enteropathogenic Yersinia spp. (Y. pseudotuberculosis
and Y. enterocolitica) and is critical for virulence. The pCD1 viru-
lence plasmid encodes the type three secretion system (T3SS) and
specific growth conditions and allows the direct injection of Yops
into the cytoplasm of target host cells, causing a variety of effects,
including modulation of the host immune response, blocking
phagocytosis, and triggering host cell death (5, 7, 15, 16, 32, 65).
Adhesins are critical virulence factors of pathogenic bacteria,
mediating interactions with host cells and allowing colonization
of specific sites within the host (55). For the pathogenic Yersinia,
adhesin-mediated host cell binding is also important to facilitate
Received 26 April 2012 Returned for modification 21 May 2012
Accepted 18 July 2012
Published ahead of print 30 July 2012
Editor: A. J. Bäumler
Address correspondence to David G. Thanassi, firstname.lastname@example.org.
*Present address: Celine Pujol, DGA Maîtrise NRBC, Le Bouchet, Vert le Petit,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
iai.asm.org Infection and Immunityp. 3490–3500 October 2012 Volume 80 Number 10
the delivery of Yop effector proteins via the pCD1-encoded T3SS.
the enteropathogenic Yersinia: YadA and Inv (18, 41, 59, 63). Al-
ternate adhesins have been identified in Y. pestis, including the
outer membrane protein Ail and the autotransporter proteins
YapC and YapE (26, 27, 46). In addition, the Y. pestis genome
contains 10 gene clusters belonging to the chaperone/usher (CU)
system present in Gram-negative bacteria that is dedicated to the
in adhesion, termed pili or fimbriae (66, 71, 77). The functions of
two of the Y. pestis CU pathways have been extensively character-
ized: the plasmid pMT1-encoded caf system that expresses the F1
capsule as described above and the psa genes coding for the pH 6
antigen. The pH 6 antigen is typically expressed at 37°C under
low-pH conditions and forms thin, flexible fibers on the bacterial
surface (48, 49). The pH 6 antigen functions in adhesion to host
cells and has been shown to bind phosphatidylcholine on lung
epithelial cells but also plays a role in evasion of phagocytosis (25,
33, 40, 49). The pH 6 antigen is a virulence factor of Y. pestis and
contributes to the pathogenesis of both bubonic and pneumonic
plague (47, 73).
Biogenesis of pili by the CU pathway relies on a periplasmic
chaperone and an integral outer membrane protein termed the
(54, 66, 71, 77). The usher genes for two of the Y. pestis CU path-
ways (y1539-1544 and y4060-4063) are disrupted by an insertion
sequence or premature stop codon (Fig. 1), and thus these path-
ways are not expected to be functional. The caf and psa genes
belong to the FGL subfamily of CU pathways that assemble thin,
flexible fibers (77). In contrast, the additional CU pathways of Y.
pestis belong to the FGS subfamily that assembles rigid, rod-like
adhesive pili. In agreement with this, Y. pestis was shown to ex-
press pilus fibers distinct from the F1 capsule and pH 6 antigen
(60). Moreover, a recent study by Felek et al. demonstrated that
heterologous expression of the six intact additional Y. pestis CU
pathways in Escherichia coli resulted in the assembly pilus-like
0352, y0561-0563, y1858-1862, and y3478-3480 enhanced biofilm
formation by E. coli (25). However, when deletion mutations of
the CU pathways were constructed in the Y. pestis KIM5 strain,
only loss of the psa locus, coding for the pH 6 antigen, resulted in
decreased adhesion to host cells and decreased biofilm formation
(25). Finally, Felek et al. found that a Y. pestis KIM5 strain con-
taining a deletion of CU pathway y1858-1862 was attenuated for
to the parental wild-type (WT) strain (25). Taken together, these
ute to virulence. However, the functions of the Y. pestis CU path-
ways in host-pathogen interactions and in the pathogenesis of
plague remain to be established.
In the present study, we tested the roles of the CU pathways of
Y. pestis in virulence, using the murine bubonic (subcutaneous)
the usher gene for each of the six intact additional CU pathways
ground. Comparison of these mutants with WT KIM5? revealed
that CU loci y0348-0352, y1858-1862, and y1869-1873 contribute
to virulence via the intranasal, but not subcutaneous, routes of
infection. We found no differences between WT Y. pestis and
usher deletion mutants for biofilm formation or autoaggregation
in vitro. However, Y. pestis KIM6? usher mutants containing de-
The caf (F1) and psa (pH 6) CU gene clusters are shown at the bottom of the figure. Usher gene y1543 is disrupted by an insertion sequence (IS). The usher for
pathway y4060-4063 is disrupted by a frameshift mutation into two open reading frames (y4061 and y4062).
Chaperone/Usher Pathways of Yersinia pestis
October 2012 Volume 80 Number 10 iai.asm.org 3491
letions in CU loci y0348-0352, y1858-1862, and y1869-1873 were
defective for adhesion to host cells compared to the WT strain.
The Y. pestis CU pathways found to function in host cell binding
sal infection, establishing a function for at least three of the addi-
tional CU pathways in Y. pestis-host interactions and the patho-
genesis of plague.
MATERIALS AND METHODS
KIM, a biovar 2.MED strain (1). Y. pestis KIM6? is an attenuated pgm-
is fully virulent and contains both the pgm locus and the pCD1 virulence
plasmid marked with an ampicillin resistance (Ampr) gene (34). Specific
growth conditions for Y. pestis are noted for each experiment. E. coli
strains were grown in LB medium at 37°C with aeration. When needed,
100 ?g/ml, kanamycin (Kan) at 50 ?g/ml, or chloramphenicol (Cml) at
topyranoside) was added to 50 ?M final concentration to induce expres-
sion of plasmid-encoded genes when necessary.
The usher genes were deleted in Y. pestis KIM6? using the pKOBEG-
sacB system (20, 22). Briefly, the pKOBEG-sacB plasmid was first intro-
duced into KIM6? by electroporation. A DNA fragment containing a
amplified by PCR with the primer pairs y(usher gene)_kanF and y(usher
gene)_kanR (Table 2). Approximately 1 to 2 ?g of purified PCR product
was used to electroporate KIM6?/pKOBEG-sacB cells. Kanrtransfor-
mants harboring the mutated allele were isolated and screened by PCR.
Plasmid pKOBEG was cured by subculturing the bacteria at 26°C on me-
(usher gene)::kan strain was then transformed with plasmid pFLP2 by
conjugation with E. coli strain S17?pir, and the Kanrcassette was excised
using the flanking FRT sites. To cure pFLP2, bacteria were subcultured at
clones. These final strains were designated KIM6? ?(usher gene) (Table
1). All usher gene deletions were confirmed by PCR using appropriate
For construction of complementing plasmids py0350, py1858, and
py1871 (Table 1), the usher genes were amplified from KIM6? by PCR
using Taq polymerase (Invitrogen) and the primer pairs listed in Table 2.
The PCR products were ligated into plasmid pGEM-T Easy (Promega),
the resulting plasmids were then digested with EcoRI and BamHI or
BamHI and SalI, and the fragments encoding the usher genes were puri-
ments were then ligated into plasmid pMMB91 that had been similarly
digested and purified. Ligation products were transformed into E. coli
genes downstream of the IPTG-inducible Ptacpromoter, were confirmed
by sequencing. Purified plasmids were then transformed into their cog-
nate KIM6? usher deletion strains.
To generate the usher deletion mutations in the fully virulent Y.
pestis KIM5? background, plasmid pCD1Ap was introduced by elec-
TABLE 1 Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s)a
Source or reference
Cloning strain; hsdR recA endA
Strain for maintenance of conditional replicative oriR? plasmids
Strain used for conjugation
Attenuated biovar 2.MED strain; pgm?, pCD1?
Deletion of usher gene y0350 in KIM6?
Deletion of usher gene y0562 in KIM6?
Deletion of usher gene y1858 in KIM6?
Deletion of usher gene y1871 in KIM6?
Deletion of usher gene y2390 in KIM6?
Deletion of usher gene y3480 in KIM6?
Fully virulent biovar 2.MED strain; KIM6?/pCD1Ap
Deletion of usher gene y0350 in KIM5?
Deletion of usher gene y0562 in KIM5?
Deletion of usher gene y1858 in KIM5?
Deletion of usher gene y1871 in KIM5?
Deletion of usher gene y2390 in KIM5?
Deletion of usher gene y3480 in KIM5?
Cloning vector; Ampr
Expression vector, IPTG-inducible promoter; Kanr
Usher gene y0350 in pMMB91
Usher gene y1858 in pMMB91
Usher gene y1871 in pMMB91
FRT sites; KanrAmpr
? phage red???, arabinose inducible; Clmr
sacB FLP-?pR; Ampr
pCD1 virulence plasmid with bla; Amprcassette
gfp3.1 in vector pMMB207, IPTG inducible; AmprClmr
aClmr, chloramphenicol resistance; Ampr, ampicillin resistance; Kanr, kanamycin resistance.
Hatkoff et al.
iai.asm.orgInfection and Immunity
troporation into the KIM6? usher deletion strains under biosafety
level 3 (BSL3) conditions. The resultant KIM5?Ap strains (Table 1)
were selected by plating onto Yersinia Selective Media (YSM) agar
containing 30 ?g/ml Amp.
Transmission electron microscopy. Y. pestis was grown at 28°C or
resuspended into phosphate-buffered saline (PBS), and then adsorbed to
were fixed with 1% glutaraldehyde for 1 min, washed twice with PBS,
tungstic acid (Ted Pella) for 35 s. The grids were examined on a TECNAI
12 BioTwin G02 microscope (FEI) at an 80-kV accelerating voltage. Dig-
ital images were captured with an AMT XR-60 charge-coupled device
digital camera system (Advanced Microscopy Techniques).
Mouse infection experiments. Mouse infections were performed at
tainment Laboratory (Newark, NJ) under BSL3 conditions. All animal
research protocols were approved by the Institutional Animal Care and
Six- to eight-week-old female C57BL/6 mice (Jackson Laboratories)
were utilized for the infections. Inoculation via the subcutaneous and
intranasal routes were used to mimic bubonic and pneumonic plague,
respectively. Y. pestis strains were grown overnight at 28°C in HIB, resus-
pended in PBS, and diluted in PBS to achieve the desired infectious dose.
TABLE 2 Primers used in this study
Primer function and nameSequence (5=–3=)
Primers used for usher deletions in Y. pestis
Primers used to confirm usher deletions in Y. pestis
Primers used to amplify usher genes for complementation plasmids
Chaperone/Usher Pathways of Yersinia pestis
October 2012 Volume 80 Number 10iai.asm.org 3493
Groups of five mice were injected subcutaneously with 50 ?l containing
200 to 250 CFU or inoculated intranasally with 25 ?l containing 2,000 to
2,500 CFU (approximately 4 to 5 50% lethal doses [LD50] for the respec-
CFU counts. The mice were observed twice daily and monitored for sur-
vival for 21 days.
Biofilm formation assay. Crystal violet staining was used to deter-
mine biofilm formation and cell attachment to polystyrene as described
by O’Toole et al. (51). Briefly, overnight cultures of Y. pestis were resus-
The OD600of the cultures was read in a microplate reader (SpectraMax).
Bacterial cultures were then washed twice with 100 ?l of PBS, and 0.01%
crystal violet was added. The bacteria were incubated with the crystal
violet for 15 min at room temperature. The bacteria were then washed
solubilized with 80% ethanol and 20% acetone. The absorbance of the
normalized to bacterial culture density.
Autoaggregation assay. Y. pestis strains were grown overnight at ei-
ther 28 or 37°C in HIB. The overnight cultures were resuspended to an
every 20 min.
Tissue culture. A549 human lung epithelial cells were grown in Dul-
becco modified Eagle medium (DMEM; Gibco) containing 10% fetal bo-
vine serum (FBS; HyClone). HEp-2 human epithelial cells were grown in
MEM (Gibco) containing 10% FBS. Murine bone marrow-derived mac-
rophages (muBMDM) were obtained as described previously (57, 58).
Briefly, cells from the femurs of female WT C57BL/6 mice were grown in
bone marrow medium (49% DMEM [Gibco], 30% L-cell supernatant,
20% FBS, 1% sodium pyruvate [Gibco]). When muBMDM were seeded
for infection, they were grown in infection medium (79% DMEM
monocyte-derived macrophages (huMDM) were isolated as described
previously (30) from healthy human donors and directly seeded at 1.5 ?
105cells/well in 24-well plates (Corning) on coverslips. The cells were
allowed to differentiate for 5 days in RPMI medium (Gibco) containing
Bacterial adhesion and invasion assays. Mammalian cells were
seeded onto coverslips in a 24-well plate (Corning) at concentrations of
1.5 ? 105cells per well, followed by incubation overnight at 37°C in 5%
or usher deletion mutant Y. pestis KIM6? strains or the same strains
at 37°C with aeration until the OD600was 0.7. Y. pestis strains containing
the pGFP plasmid (for invasion studies) or deletion strains containing
complementation plasmids were induced for expression of the plasmid-
encoded genes by the addition of IPTG at 2 h of growth. For adhesion
experiments, the bacteria were resuspended to a multiplicity of infection
(MOI) of 50 in the appropriate medium for the host cell type used. For
invasion experiments, bacteria were resuspended to an MOI of 50 for the
epithelial cells or an MOI of 10 for the macrophages. For adhesion exper-
iments, the muBMDM and huMDM were pretreated with 5 ?g of cy-
the plate was centrifuged (50 ? g, 4 min, room temperature) to facilitate
bacterial contact. After 2 h at 37°C in 5% CO2(or 20 min for the macro-
30 min in 2.5% paraformaldehyde at room temperature. All subsequent
steps were completed at room temperature. The cells were blocked with
3% bovine serum albumin (BSA) in PBS for 20 min and then incubated
with rabbit anti-Yersinia antiserum SB349 (6) diluted 1:1,000 in 3% BSA
a secondary goat anti-rabbit antibody conjugated to Alexa Fluor 594 (In-
vitrogen) was added at a 1:2,000 dilution in 3% BSA in PBS, followed by
incubation for 30 min. The cells were then washed again three times with
PBS. The coverslips were mounted on glass slides using ProLong Gold
antifade reagent (Invitrogen), and the slides were examined on a Zeiss
Axioplan2 microscope using a ?40 objective lens. Phase-contrast and
epifluorescence images were captured using a Spot camera (Diagnostic
Instruments) and processed using Adobe Photoshop. For each experi-
the host cells were quantified for each field to calculate the number of
bacteria/cell. The values obtained for the 10 fields were then averaged to
obtain the number of bacteria/cell for the experimental replicate.
Bacterial survival during macrophage infection. muBMDM were
seeded on coverslips in a 24-well plate (Corning) at concentrations of
1.5 ? 105cells per well, followed by incubation overnight at 37°C in 5%
or usher deletion mutant Y. pestis KIM6? strains were diluted 1:20 into
fresh HIB and grown at 37°C with aeration until the OD600was 0.7. The
muBMDM were washed three times with PBS, 1 ml of bacteria with an
MOI of 5 was added to each well, and the plate was centrifuged (50 ? g, 4
min, room temperature) to facilitate bacterial contact. After 20 min at
37°C, 5% CO2the cells were washed once with PBS, and fresh infection
three times with 1 ml of PBS. Fresh medium with or without 2 ?g of
were incubated for an additional 23 h.
in PBS for 10 min at 37°C. The lysates were removed, the wells were
washed with 0.5 ml of PBS, and the lysates and wash were pooled in a
1.5-ml microcentrifuge tube. Serial 10-fold dilutions were then plated on
LB agar, followed by incubation at 28°C for 2 days, and the CFU were
Outer membrane isolation and analysis. KIM6? WT or the usher
at 37°C with aeration until an OD600of ?0.7 was reached. Bacteria were
harvested, washed, resuspended in 1 ml of 20 mM Tris-HCl (pH 8) con-
taining Complete protease inhibitor cocktail (Roche), and lysed by soni-
cation for 2 min (15 s on, 15 s off) in an ice-water bath. Whole bacteria
were removed by centrifugation (8,000 ? g, 2 min, 4°C). Sarkosyl (sodi-
um-N-lauroylsarcosinate; Fisher) was added to the supernatant fraction
room temperature to selectively solubilize the cytoplasmic membrane.
The outer membrane was then pelleted by centrifugation (15,000 ? g, 30
min, 4°C) and resuspended in 0.1 ml of 20 mM Tris (pH 8)–0.3 M NaCl.
An equal volume of 2? sodium dodecyl sulfate (SDS) sample buffer was
added, and the sample was incubated for 10 min at 95°C prior to separa-
tion by SDS-polyacrylamide gel electrophoresis (PAGE). The expression
massie blue-stained SDS-PAGE gels or by immunoblotting with anti-F1
antibody (60) at 1:1,000, anti-Pla antibody (72) at 1:500, anti-PsaA anti-
body (47) at 1:1,500, or anti-Ail antibody (76) at 1:500. Immunoblots
were developed with alkaline phosphatase-conjugated secondary anti-
bodies and BCIP (5-bromo-4-chloro-3-indolylphosphate)/NBT (ni-
troblue tetrazolium) substrate (KPL).
Statistical analysis. Mouse survival curves were compared using the
log-rank test with data obtained from three independent experiments
to six independent experiments with three replicates each. Statistical sig-
tiple-comparison post test. Statistical calculations were performed using
Hatkoff et al.
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